This invention relates to a high efficiency chromatographic system. More specifically, the present invention relates to a chromatographic system for determining the physicochemical properties of one or more compounds using at least two chromatographic units in eluent flow communication with one eluent analyzer via an intermediate eluent switch.
The emergence of automated chemical synthesis platforms coupled with combinatorial techniques as a routine tool in the pharmaceutical industry has enabled the synthesis of large numbers of molecules in a relatively short time. Millions of potential new drug candidates are created every year, and both pharmaceutical and biotechnology industries have embraced the challenge in recent years of developing new, faster and more efficient ways to screen pharmaceutical compounds in order to rapidly identify xe2x80x9chitsxe2x80x9d and develop them into promising lead candidates. This has created the need for high-throughput analytical approaches to characterize the synthesized compounds and has prompted the development of chromatographic systems specifically designed for the automated high-throughput identification, purity assessment or purification of combinatorial libraries.
Currently, automated, semi-quantitative assessment of combinatorial libraries is most readily accomplished by coupling HPLC with UV detection and mass spectrometry. Rapid HPLC methods with columns capable of delivering high-resolution separations have been developed in recent years, and have been well received by the drug discovery industry as a powerful tool particularly suited to handle the expanding analytical needs of combinatorial chemistry. The ability to characterize chemical libraries derived from combinatorial synthesis has in turn revealed that the purity of the compounds generated by this method is not necessarily high enough for biological evaluation of these compounds. Consequently, the scope of the high-throughput HPLC techniques initially designed and developed for structure confirmation purposes has expanded to include purity assessment and purification of the compound libraries to make them suitable for biological screening.
The technological advances directed toward the implementation of fast and high volume chromatographic systems have rapidly converged toward automated systems to accommodate the large number of compounds typically produced by most parallel syntheses nitially, automated preparative HPLC systems were designed so as to incorporate a fraction collection device activated upon detection of a threshold UV signal operating in conjunction with a secondary analytical unit (e.g., flow injection MS HPLC-ESI-MS) for the identification of the collected fractions. Recently, Kassel et al. (Zeng L., Burton L., Yung K., Shushan B., Kassel D. B., xe2x80x9cAutomated Analytical/Preparative High-Performance Liquid Chromnatography-Mass Spectrometry System for the Rapid Characterization and Purification of Compound Librariesxe2x80x9d, J. Chrom. A, 794, 3-13, (1998)) added a major improvement to the technology by incorporating a xe2x80x9cspecific-mass-basedxe2x80x9d fraction collection device: fraction collection is initiated upon a real-time threshold reconstructed ion current signal being observed for a particular m/z input value, which corresponds to the mass of the compound being purified. This eliminates the need for post-purification screening and pooling required to identify the purified fractions of interest. Finally, they developed the system further and conceived an improved version of it (xe2x80x9cDevelopment of a Fully Automated HPLC/Mass Spectrometry System for the Analytical Characterization and Preparative Purification of Coinbinatorial Librariesxe2x80x9d, Anal. Chem., 70(20), 4380-4388, (1998)). Kassel and coworkers devised an automated parallel analytical/preparative LC/MS system incorporating fast reversed-phase HPLC and electrospray ionization mass spectroscopy (ESI-MS), capable of processing the purification of two 96-well microtiter plates in parallel.
The system designed by Kassel et al. is comprised of two identical columns (analytical or preparative) running in parallel and is interfaced with two 96-well microtiter plates, each well containing a single synthetic product. Incorporation of a switching valve permits sequential loading of the samples onto the two columns: the autosampler draws the content of the first well of microtiter plate 1 and injects it onto the first column, then the autosampler picks up the content of the first well of microtiter plate 2 and loads it onto the second column. The same mobile phase is delivered to each column from a single HPLC pumping system, the flow from the pump splitting evenly between the columns (provided that the columns have comparable back pressures). Kassel et al. modified the IonSpray interface of the system to support flows from multiple columns and the eluents of the two columns were simultaneously introduced into the IonSpray source housing, and analyzed by mass spectrometry. This particular configuration allows the purification of chemical libraries based on mass spectrometry signal-detected fraction collection. Prior to performing the chromatographic separation, the mass and position of the expected products synthesized in the microtiter plate wells are specified. When a particular compound is detected by mass spectrometry in the course of the HPLC elution, the fraction collector connected to the column from which the compound is eluting is triggered, and the sample is collected in a specific tube determined by the position of the autosampler (for example, if the sample is drawn from well 1 of the autosampler/synthesis rack, the sample will be collected into tube 1 of the fraction collector rack). Thus, only compounds matching the molecular weight of the desired products are collected, and only one fraction is collected for each sample injected.
A major limitation of Kassel et al.""s parallel LC/MS technique is that the products to analyze must be of unique mass: false triggering of the fraction collectors is observed if two eluted compounds are of the same mass and similar ionization response. Thus, the synthesis of the combinatorial libraries must be carried out with the added restriction that no two expected products should yield the same molecular weight products. Further, for the flow to be equivalently transferred to the two columns requires that they have comparable back pressures because delivery of the solvent gradient is performed by a single pumping system. This generally requires that the columns be of the same size and be packed with the same chromatographic material. In addition, the design also dictates that both columns are eluted with identical mobile phase compositions. This limitation is usually of no consequence for the purification/purity assessment of combinatorial libraries, since the synthesized compounds are generally structurally related and exhibit similar chromatographic behaviors.
The Kassel et al. system is particularly well suited for one of the major challenges found in the pharmaceutical industry: high-throughput structure confirmation and purity evaluation of large numbers of compounds derived from combinatorial syntheses. However, it does not address the other essential aspect of the drug discovery process: the physicochemical characterization of large numbers of compounds derived from parallel synthesis for quantitative structure-activity relationships (QSAR) studies, and the implementation of massive screening techniques for the biological evaluation of compound libraries.
Micromass(copyright) (Manchester, UK) implemented a multiplexed electrospray interface that is capable of sampling four individual liquid streams in rapid succession. The system comprises a single pump delivering solvent to all four columns run in parallel. The system has been integrated with the Z-Spray ion source of the Micromass(copyright) LCT orthogonal acceleration time-of-flight mass spectrometer. The inner source housing contains an array of four pneumatically assisted electrospray probe tips that are directed at the sampling cone. A hollow cylinder is positioned co-axially with the sampling cone. Two diametrically opposed circular apertures in the wall of the cylinder allow the spray from one electrospray probe tip to pass through the cylinder across the sampling cone, while all the other sprays are excluded. The spray from each probe tip is admitted in turn to the sampling cone as the cylinder is rotated by a programmable stepper motor. The source is supplied with a heated stream of dry nitrogen that facilitates the desolvation of ions in the selected stream. To monitor the four separate electrosprays, the rotor is rotated from position to position. An optical encoder indicates which spray channel is being sampled at any one time, and the data from that channel is written to its own specific data file. The system has recently been upgraded to include a total of 8 channels.
One of the disadvantages of this system is that is uses a single solvent delivery system for all the columns. Thus, the user is restricted to use column of identical size and packing material if the mobile phase flow is to be split equally through each column. In addition, the system utilizes a proprietary dual orthogonal xe2x80x9cZxe2x80x9d sampling technique, which cannot be readily adapted to other mass spectrometers, much less to other types of detectors. Furthermore, mobile phase is continuously eluting from all the LC channels/probe tips in operation during any given run. The flow from the spray tips is never interrupted. The inner source cylinder functions essentially as a screen for all the sprays but one. The eluent from the sprays that are denied access to the MS capillary inlet thus hits the outside wall of the inner source cylinder. The stream of heated dry nitrogen gas facilitates evaporation of the solvent in the atmosphere. Although the maximum flow from each electrospray channel is small (100 xcexcL/min) this potentially constitutes a health hazard, depending on the nature of the mobile phase or the analyte.
With the advent of combinatorial chemistry and the need to develop assays for the large numbers of compounds being made available using that technology, many researchers have focused their efforts on developing in vitro tests/assays that provide biologically significant compound information. Much work has been directed to the correlation of certain physicochemical properties with biological activity, both in the search for new therapeutic agents and in the understanding of compound toxicity from medicinal and environmental perspectives. For example, physicochemical properties of recognized significance to evaluation of a compound""s biological activity are its lipophilicity, hydrophilicity, interfacial pKa, and membrane affinity, among others. The determination of these properties is critical for QSAR studies, and the worldwide discovery effort. The present invention relates to a system for determining not only chemical structure, but also the physicochemical properties critical for such QSAR studies and drug discovery efforts.
The chromatographic process represents a reversible equilibrium of solutes between the mobile phases and the stationary phases. The magnitude of solute retention is a direct result from this equilibrium and is typically expressed by a parameter, the capacity factor, kxe2x80x2=(trxe2x88x92to)/to where to is the dead time and tr is the retention time of the solutes. The capacity factor is therefore a stoichiometric mass distribution equilibrium of solutes between the mobile phases and the stationary phases, and its determination allows the calculation of various physicochemical values according to pre-determined algorithms.
The distinction between serial and parallel column chromatography is important. Serial column chromatography is an established method and involves automatically changing columns after a chromatographic run. This allows multiple columns to sequentially be evaluated. In contrast, parallel column chromatography allows multiple columns to simultaneously access one detector. Parallel chromatography has intrinsically higher throughput compared to serial chromatography.
Reducing the costs of analyzing large numbers of compounds is a commercial driving force for developing parallel chromatography systems. Chemical and biological studies that require the analysis of large numbers of samples have become routine for both drug discovery and drug analysis. Applications include routine chemical analysis, clinical trial samples, purification of compounds, obtaining physical or chemical parameters, measuring membrane binding properties, analyzing the quality of chemical libraries, natural product screening, chiral chromatography, etc.
These analysis problems are particularly amenable to parallel chromatography. For instance, high throughput screening of chemical libraries in drug discovery typically requires evaluation of thousands of samples. Similarly the analysis of biological samples from clinical trial studies can involve the analysis of large numbers of compounds and it is not uncommon to have  greater than 5000 samples for analysis in clinical trials.
The present invention finds application in chromatographic analyses of large numbers of compounds exhibiting actual or potential activity of biological or clinical significance. In such analyses, compounds are characterized and compared to compounds of known activity by their relative affinities to multiple stationary phases of biological significance, for example, using high performance liquid chromatography columns. Of particular use as stationary phases in such procedures are immobilized artificial membranes such as those described and claimed in U.S. Pat. No. 4,931,498. One approach to such analytical procedures is described and claimed in PCT patent application serial no. PCT/US98/17398, published Mar. 4, 1999, as WO99/10522, the content of which is incorporated herein by reference.
The present chromatographic system allows determination of physicochemical properties through the use of multiple chromatographic units in communication with one eluent analyzer via an eluent switch. The present invention dramatically increases the performance, efficiency, scope of use and commercial value of said chromatographic unit/eluent analyzer system. The chromatographic unit can be any chromatographic system that can be interfaced with an analyzer or detector, and can include (but is not limited to) high-performance liquid chromatography (HPLC) columns, capillary electrophoresis chromatography (CEC) columns, Gas Chromatography (GC) columns, super-critical fluid columns and microchips. The eluent analyzer unit is any instrument capable of identifying the presence, physicochemical characteristics and/or chemical structure of a compound, including (but not limited to) a mass spectrometer (MS), a Fourier transform infra red spectrometer (FTIR), a Fourier transform ultra violet spectrometer (FTUV), standard UV detector, fluorescent detector, electrochemical detector, refractive index detector and a Fourier transform nuclear magnetic resonance spectrometer (FTNMR).